Macroscopic IK[Ca is not expressed in normal levels in chick ciliary ganglion (CG) neurons prior to synapse formation with target tissues, or in neurons developing in vitro or in situ in the absence of target tissues. Here, two chick CG slo partial cDNAs encoding IK[Ca channels were isolated, cloned, and sequenced. Both slo transcripts were readily detected in developing CG neurons prior to or in the absence of target tissue interactions. When CG neurons developed in vitro in the presence of target tissue (iris) extracts, a normal whole-cell IK[Ca was expressed. These effects did not require protein synthesis, and the activity was detectable throughout the stages of synapse formation in the iris. The active component has an apparent molecular weight of 40-60 kDa.
1. The functional expression of CaP-activated K+ currents (IK(Ca)) and voltage-activated Ca2+ currents (ICa) was examined using whole-cell recordings from chick lumbar sympathetic neurones developing in situ and under various conditions in vitro.2. Macroscopic IK(ca) was expressed at low current density (<0 01 mA cm-2) in neurones isolated at embryonic days 9-16 (E9-16). IK(ca) was expressed at high densities (>0 04 mA cm-2) at El 7-19. By contrast, there was no significant difference in ICa density between sympathetic neurones isolated at E13 and E18. 3. When sympathetic neurones were isolated at E13 and maintained in vitro for 5 days, IK (Ca) was expressed at a significantly lower density (< 0 01 mA cm-2) than in neurones isolated acutely at E18 (>0 04 mA cm-2). There was no difference in ICa density between neurones that developed in vitro and in situ. 4. When El 3 sympathetic neurones were cultured for 5 days in the presence of a confluent layer of ventricular myocytes, they expressed IK(Ca) at a high density (>0 04 mA cm-2), similar to that of E18 neurones that developed entirely in situ. Cardiac cell-conditioned medium produced similar effects. However, co-culture of sympathetic neurones with spinal cord explants did not allow for normal IK(ca) expression in vitro. 5. Culturing sympathetic neurones in the presence of 5 ng ml-' nerve growth factor (NGF) caused a significant increase in IK(ca) density but this effect was only seen in 50% of cells examined.6. The largest developmental changes in macroscopic IK(ca) occur several days after other K+ currents and ICa are expressed at maximal density. The normal developmental expression of IK(Ca) is dependent upon extrinsic factors, including target-derived differentiation factors. Different populations of neurones express differentensembles of voltage-and Ca2+-activated ion channels.Differences in the expression of ion channels have important functional consequences as they allow neurones to express a specific electrophysiological phenotype suitable for the appropriate processing of information. Moreover, ion channels play an important role in the development and differentiation of vertebrate neurones, including regulation of the dependence of neurones on neurotrophic factors, neurite outgrowth and guidance, and the expression of neurotransmitters and their metabolic enzymes (reviewed by Spitzer, 1991;Spitzer, Gu & Olson, 1994 Spitzer, 1991;Ribera & Spitzer, 1992). Instead, the various ion channels appear gradually at developmental stages, and in a sequence, specific for a given cell type. In many populations of vertebrate neurones, those ion channels that produce subtle effects on action potential waveform and in the patterns of action potential discharge are expressed later than those channels that are essential for the minimal manifestation of
1. The functional expression of transient voltage-activated K+ currents (IA) was examined using whole-cell recording techniques in embryonic chick sympathetic ganglion neurones that developed in situ and under various growth conditions in vitro. 2. The density of IA increased dramatically during development in sympathetic neurones isolated acutely between embryonic days 7 and 20 (E7-E20). The time course of IA inactivation became significantly faster between E7 and E13. With these protocols, neuronal differentiation and development occurred entirely in situ. 3. Sympathetic neurones isolated at E9 and maintained in vitro for 4 days did not express a normal IA compared to neurones isolated acutely at E13. Those neurones that were in physical contact with other neurones expressed normal densities of IA, but the resulting inactivation kinetics were abnormally slow. Sympathetic neurones that were cultured on the membrane fragments of lysed neurones expressed normal densities of IA even when they failed to make visible connections with other viable neurones, but the resulting inactivation kinetics were abnormally slow. Those cultured neurones that were not in physical contact with other cells or their membranes had markedly reduced
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